Background: Milking frequency influences milk production in cows by regulating milk synthesis and secretion (i). The higher or lower milk synthesis and secretion involve a number of genes that are differentially expressed. It has been well established that variation in DNA methylation at the CpGs located in the regulatory region of genes causes variation in gene expression. In this study, we hypothesized that milking frequency changes the milk yield through altered gene expression by modifying the methylation at CpGs dinucleotides located in gene regulatory regions. Materials & Methods: Eight heifers were studied in three periods. Period 1: twice daily milking for 5 days: both udders of each heifer were milked twice a day. Period 2: differential milking for 7 days: right udder was milked twice a day; left udder was milked once a day. Period 3: both udders returned to twice daily milking. Milk yield was recorded every day over the three periods. Biopsies from left & right udders were taken at the end of period 2 for histology, DNA, and RNA preparations. Global DNA methylation was evaluated using the LUMA assay. DNA methylation of three CpGs located within a distal regulatory region of one casein gene, CSN1, was analyzed using pyrosequencing of the PCR products amplified from bisulphite treated genomic DNA. The genome wide methylated regions will be captured and sequenced using MeDIP-Seq. The mRNA expression of CSN1 and other candidate genes obtained from MeDIP-Seq will be quantified using RT-qPCR. Results: Global methylation of the once daily milking group (75.9%) is higher compared to the twice daily milking group (74.6%). However the difference is not statistically significant (P=0.16). A closer look at the global methylation of each individual showed that one of eight heifers (8156) behaved differently from the others. When this individual was excluded, the two sets of data became significantly different (P=0.04). The DNA methylation of all three CpG1, CpG2, and CpG3 of once daily milking group is higher than that of twice daily milking group (P=0.02, P=0.07, and P=0.02, respectively). Those results are in accordance with global methylation profiles. Conclusion: Milking frequency modifies the DNA methylation to a certain extent at the global level and at the local CSN1 regulatory region in the bovine mammary gland. We are presently studying the relation of those modified DNA methylation and CSN1 mRNA expression using RT-qPCR. At the genome wide level, all possible methylated regulatory regions in the genome will be captured and sequenced using MEDIP-Seq. Gene expression of corresponding candidate genes will then be quantified. (i) Boutinaud et al. 2008. J Dairy Sci. 91: 988-98.